The intracellular movement of viral particles requires directed motor-based transport on long-range microtubule (MT) networks. However, the molecular details by which these dynamic host networks function during infection remain poorly understood. Members of this Program Project Grant (PPG) have observed diverse changes in MT organization and stability during infection with distinct RNA or DNA viruses. In addition, we have identified roles for specialized MT regulatory factors in mediating MT rearrangements and viral infection as well as key motors involved in Adenovirus bi- directional motility. As part of our Overall Aims to develop a truly detailed understanding of how these motile invaders interact with and exploit the equally dynamic MT networks of their host over the course of infection, we will establish the Imaging Core. Run by an expert in live imaging of organelle and virus motility (Dr. Richard Vallee), the Imaging Core will provide state of the art high temporal and high spatial resolution dual-fluorescence live imaging to support the individual Aims of each of the five members of this PPG. The capacity of this equipment will allow real-time tracking of viral particle movements and their interactions with host MTs at various stages of infection in living cells. This imaging system will also be used to develop new ways of visualizing stable MTs in living cells that should allow for the first time direct tests of trafficking of cargos on specific MT subtypes, as well as developing new ways of generating post-translationally modified MTs in vivo to define viral and motor protein interactions with specific MT subsets. We will also test the effects of plus- end tracking MT regulators on MT dynamics and stabilization and their influence on virion movement in living cells, as well as imaging the effects of infection on MT plus-end tracking protein activity. This will provide an unprecedented level of analysis of these highly dynamic processes and the role of specialized MT regulatory factors and motors, using novel approaches to explore the potential for selective use of dynamic MT capture versus trafficking on stable MT subsets by distinct viruses. To this end, the Imaging Core will provide a central state of the art resource to each PPG member and which plays a fundamental role in addressing the Overall Aims of this PPG to understand MT networks and virus trafficking in diverse viral systems.